Dan Zheng

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed. Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions. Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated. Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers. Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance.

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal–Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.